Analysis of the functional divergence of the Notch ligands Delta1 and Delta4 in vitro and in vivo

进化保守的Notch信号通路介导相邻细胞之间的直接通讯，对线虫、昆虫和脊椎动物等多种生物的多种发育过程的调控起着关键作用。DLL1和DLL4是两个哺乳动物DSL蛋白，它们的氨基酸序列相似(47%相同，14%相似氨基酸)和结构域结构(都包含相同数量的EGF样重复序列，以及C端的PDZ结合域)，都具有激活Notch配体的功能。这些配体在发育过程中和成年组织中以独特和重叠的模式表达。在肠上皮中，DLL1和DLL4在隐窝细胞中共表达，并在维持隐窝祖细胞库方面起冗余作用。在胸腺中，DLL1和DLL4均在胸腺上皮细胞中表达，基质细胞上表达的DLL1和DLL4都能在体外诱导造血祖细胞的T细胞发育。然而，在体内，DLL4是T淋巴细胞生成所必需的Notch配体。DLL4能更好地结合胸腺细胞上的受体，并且需要较低的稳定细胞表面水平来诱导T细胞体外发育。与这些观察一致的是，用纯化的DLL1和DLL4片段以及NOTCH1进行的生化研究表明，DLL4与NOTCH1结合的亲和力大约是NOTCH1的10倍。出人意料的是，我们使用两个小鼠模型的初步数据显示-尽管DLL4具有更强的Notch激活潜力-在胚胎发育早期，DLL4不能取代DLL1s在体细胞发生和肌肉分化中的功能，这表明这些密切相关的DSL蛋白的生化和功能特性存在着深刻的差异。在这项研究中，我们建议检验这一假设，即DLL1和DLL4的特定蛋白结构域赋予这些配体不同的生化性质，从而导致非冗余和冗余功能依赖于上下文。这将通过分析嵌合的DLL1/4配体蛋白来实现，这些蛋白的结构域已经被交换。嵌合配体将在体外分析其Notch激活潜力和生化特性，以及在体内胚胎发育期间使用已建立的转基因策略。这些实验将阐明Notch配体DLL1和DLL4的不同生化特性，这是两种密切相关的DSL蛋白激活Notch的不同结果的基础，并应提供新的见解，了解这一至关重要的信号通路如何在体内激活并根据上下文进行微调。

The evolutionary conserved Notch signaling pathway mediates the direct communication between adjacent cells, and is pivotal for the regulation of multiple developmental processes in diverse organisms such as nematodes, insects and vertebrates. DLL1 and DLL4 are two mammalian DSL proteins that are similar in their amino acid sequence (47% identical plus 14% similar amino acids) and domain structure (both contain the same number of EGF-like repeats, and a C-terminal PDZ binding domain) and both function as activating Notch ligands. These ligands are expressed both in unique and overlapping patterns during development and in adult tissues. In the intestinal epithelium DLL1 and DLL4 are coexpressed in crypt cells and act redundantly in maintaining the crypt progenitor cell pool. In the thymus DLL1 and DLL4 are both expressed in thymic epithelial cells, and both ligands expressed on stromal cells can induce T cell development of hematopoietic progenitors in vitro. However, in vivo DLL4 is the essential Notch ligand required for T-lymphopoiesis. DLL4 binds better to NOTCH receptors on thymocytes and a lower steady-state cell surface levels is required to induce T-cell development in vitro. Consistent with these observations biochemical studies with purified fragments of DLL1 and DLL4 and NOTCH1 have shown that DLL4 binds to NOTCH1 with approximately 10-fold higher affinity.Unexpectedly, our preliminary data using two mouse models show that -despite the stronger Notch activation potential of DLL4- during early embryonic development DLL4 cannot replace DLL1s function during somitogenesis and muscle differentiation, suggesting profound differences in the biochemical and functional properties of these closely related DSL proteins. In this study we propose to test the hypothesis that specific protein domains of DLL1 and DLL4 confer different biochemical properties to these ligands and thereby lead to non-redundant and redundant functions depending on the context. This will be achieved by the analysis of chimeric DLL1/4 ligand proteins in which domains of these proteins have been swapped. Chimeric ligands will be analyzed in vitro for their Notch activation potential and biochemical properties as well as during embryonic development in vivo using an established transgenic strategy. These experiments should elucidate the divergent biochemical properties of the Notch ligands DLL1 and DLL4 that are the basis for the different outcomes of NOTCH activation by two closely related DSL proteins and should provide novel insights how this critically important signaling pathway is activated and fine-tuned in vivo depending on the context.